E. coli mismatch repair enhances AT-to-GC mutagenesis caused by alkylating agents.

Alkylating agents are known to induce the formation of O6 -alkylguanine (O6 -alkG) and O4 -alkylthymine (O4 -alkT) in DNA. These lesions have been widely investigated as major sources of mutations. We previously showed that mismatch repair (MMR) facilitates the suppression of GC-to-AT mutations caused by O6 -methylguanine more efficiently than the suppression of GC-to-AT mutations caused by O6 -ethylguanine. However, the manner by which O4 -alkyT lesions are repaired remains unclear. In the present study, we investigated the repair pathway involved in the repair of O4 -alkT. The E. coli CC106 strain, which harbors Δprolac in its genomic DNA and carries the F'CC106 episome, can be used to detect AT-to-GC reverse-mutation of the gene encoding β-galactosidase. Such AT-to-GC mutations should be induced through the formation of O4 -alkT at AT base pairs. As expected, an O6 -alkylguanine-DNA alkyltransferase (AGT) -deficient CC106 strain, which is defective in both ada and agt genes, exhibited elevated mutant frequencies in the presence of methylating agents and ethylating agents. However, in the UvrA-deficient strain, the methylating agents were less mutagenic than in wild-type, while ethylating agents were more mutagenic than in wild-type, as observed with agents that induce O6 -alkylguanine modifications. Unexpectedly, the mutant frequencies decreased in a MutS-deficient strain, and a similar tendency was observed in MutL- or MutH-deficient strains. Thus, MMR appears to promote mutation at AT base pairs. Similar results were obtained in experiments employing double-mutant strains harboring defects in both MMR and AGT, or MMR and NER. E. coli MMR enhances AT-to-GC mutagenesis, such as that caused by O4 -alkylthymine. We hypothesize that the MutS protein recognizes the O4 -alkT:A base pair more efficiently than O4 -alkT:G. Such a distinction would result in misincorporation of G at the O4 -alkT site, followed by higher mutation frequencies in wild-type cells, which have MutS protein, compared to MMR-deficient strains.